The present invention relates to novel compounds of use in treatment of methicillin resistant Staphylococcus aureus (MRSA) infection in conjunction with β-lactam antibiotics.
S. aureus is one of the major causes of both nosocomial and community-acquired infections worldwide. The use and overuse of β-lactam agents and other antibiotics has resulted in intense selective pressure on bacterial populations and led to the emergence of multi-drug resistant bacteria that threaten our ability to treat serious infections, particularly in hospitals. Over the last 15 years there has been a steady rise in the incidence of methicillin-resistant S. aureus (MRSA) and the latest figures from the PHLS Communicable Disease Surveillance Centre indicate that, in England and Wales, about 45% of S. aureus isolates are now resistant to this agent. Staphylococci show a strong tendency to accumulate antibiotic resistant genes and the majority of MRSA isolates are now resistant to a range of antibiotics. Ominously, MRSA strains carrying the enterococcal vanA gene complex and expressing high-level resistance to vancomycin have recently been identified in clinical specimens from two unrelated cases. The introduction of Synercid and Linezolid and the anticipation of a third agent with activity against MRSA (Daptomycin) supplements the anti-MRSA armamentarium but there remains an urgent need for new treatments for these infections, in particular agents that suppress or abrogate the emergence of resistance. We are examining the therapeutic potential of agents that do not directly kill the target bacterial population but modify them to produce a “less fit” phenotype with reduced capacity to survive at the site of infection. There are conceptual reasons to suppose that this approach will result in less selective pressure on the bacteria and delay the emergence of resistant genotypes.
Polyphenolic components extracted from Japanese green tea (Camellia sinensis) possessed a number of activities against methicillin resistant S. aureus (MRSA) (Yam, T. S. et al 1997; Yam, T. S. et al 1998; Stapleton, P. D. et al (2002)) in addition to weak direct antibacterial activity, extracts were able to suppress the activity of staphylococcal β-lactamases. In addition, at subinhibitory concentrations they were also able to sensitise MRSA strains to methicillin and other semi-synthetic β-lactam antibiotics; this effect was marked and reduced the Minimum Inhibitory Concentration (MIC) of test strains from full resistance to below the antibiotic break point.
Initial observations were made using aqueous extracts of green tea; partition chromatography was then used to fractionate the material (Yam, T. S. et al. 1997). Activity was confined to one fraction that was enriched for the compound epigallocatechin gallate ECg, an abundant polyphenolic component of green tea, but other constituents were present in small amounts.
Unfortunately, epicatechin gallate cannot be widely administered because it is broken down by esterases in the body to the inactive products, epicatechin and gallic acid (Kohri, T. et al, 2001).
It has been observed that MRSA grown in the presence of sub-inhibitory concentrations of tea extracts have thickened cell walls and form pseudomulticellular aggregates (Hamilton-Miller, J. M. T et al (1999)). Green tea administered as a spray has been successfully used in the treatment of an MRSA infection of the trachea (windpipe) (Yamashita, S. et al (2001)).
Stapleton et al. (2004) describe some investigations into the molecular mechanism of β-lactam sensitisation to establish a basis for the rational selection of pharmacologically acceptable molecules, investigated structure-activity relationships (SAR) to identify pharmacophores within active molecules. The capacity of catechins and gallates to modulate β-lactam resistance was evaluated by testing the compound at a fixed concentration in combination with oxacillin. Modulating activity was defined as a greater than two-fold reduction in the MIC of a β-lactam when tested in combination with a fixed sub-inhibitory concentration of the test compound. ECg converted MRSA strain BB568, which has a MIC of 256 mg/L for oxacillin, to the fully drug sensitive phenotype (1 mg/L) and below the breakpoint. EGCg reduced the MIC of BB568 to 8 mg/L; epicatechin (EC) and epigallocatechin (EGC), the two most abundant non-gallyl catechins in tea, were inactive in the combination assay. These compounds were tested against a comprehensive collection of 40 MRSA strains isolated from a variety of countries; epidemic MRSA strains from the UK were included. With all strains, ECg reduced the MIC values to the susceptibility breakpoint or below.
The physical properties of ECg and EGCg suggest these molecules have the capacity to intercalate into target membrane bilayers and perturb the function of key membrane-associated proteins in peptidoglycan synthesis, such as femA, femB and mecA gene products; it is highly likely that such an interaction would also reduce or prevent the transport of proteins not essential for cell viability across the bilayer. Japanese workers have shown that catechins are able to bind to artificial lipid bilayers (Nakayama, T. et al 1998, Hashimoto, T. et al 1999) and recent work (Kajiya, K. et al 2001 and Kajiya, K. et al 2002) shows that binding affinities appear to correlate with the bioactivity of catechins in our assay. Thus, ECg has a greater propensity to intercalate into the phospholipid palisade than EGCg, catechin or EC (Cartula, N. et al 2003). Binding has been shown to be dependent upon the number of hydroxyl groups on the B-ring catechins with two hydroxyl groups, such as ECg, have a greater membrane binding capacity than those with three hydroxyl groups, such as EGCg (Kajiya, K. et al 2001). The binding of ECg to liposomes is enhanced in the presence of EC (Kajiya, K. et al 2002).
Surprisingly, it has been found that intercalation of catechin gallates into the cytoplasmic membrane interferes with the export of proteins such as—toxin and coagulase and raises the possibility that the compounds may reduce the virulence of Gram-positive bacteria such as S. aureus at the site of infection and thus significantly contribute to their removal from the body by immune processes (Taylor, P. et al. 2004).